ABSTRACT Hyposalivation, which leads to ?dry mouth? and complications thereof, is a debilitating effect of Sjogren's Syndrome and occurs as a side-effect of radiation treatment for head and neck cancers. Significant alterations in the vasculature correlate with salivary hypofunction. Microvessel density is decreased in Sjogren's Syndrome and in irradiated glands, and aberrant vessels and premature arteriosclerosis are reported in Sjogren's Syndrome. Post-irradiation transplantation of bone marrow-derived and adipose-derived mesenchymal stem cells (MSCs) improves salivary flow and microvessel density. Additionally, transient delivery of FGF2 and VEGF after radiation damage restores microvessel density and salivary flow. Despite this evidence for restoration of vascular function as a treatment for salivary hypofunction, there are currently no therapies available to stimulate normal vessel structure and function in diseased salivary glands. The ability of combined therapies of endothelial cells (ECs) and MSCs to stimulate regeneration has not been directly tested. Further, the mechanisms through which ECs and MSCs restore salivary function remain unclear. Vasculature, which is comprised of ECs and pericyte support cells of mesenchymal origin, is known to perfuse tissues with nutrients. Additionally, ECs residing in capillaries are known to create vascular niches that stimulate regeneration in many organs through paracrine- and juxtacrine-acting factors that function as stem and progenitor cell-active trophogens. The profile of paracrine-acting factors produced by tissue-resident ECs is known to be organ-specific but remains uncharacterized in salivary glands. With this proposal we will address the following questions: 1) Can ECs stimulate salivary gland regeneration in vivo? 2) Is the ability of ECs to stimulate regeneration enhanced by MSCs? 3) What EC-dependent factors stimulate salivary gland regeneration? Here we investigate the hypothesis that salivary ECs secrete paracrine factors that stimulate functional salivary gland regeneration. To determine if EC +/- MSC transplantation enhances regeneration, in Aim 1 we will deliver EC +/- MSCs via hydrogels into a salivary gland resection model and examine gland regeneration at a cellular level and gland function by measuring salivary flow. The matrix metalloproteinase-degradable poly(ethylene glycol) (PEG)-based hydrogels have been used to successfully deliver MSCs into bone defect models in vivo. To identify paracrine-acting factors produced by ECs that could be used therapeutically, in Aim 2 we will use next generation RNA sequencing (RNA seq) to identify potential EC-dependent therapeutic factors and validate them in co-culture assays. This study will establish a new research team who will deliver proof of concept data for EC/MSC-dependent regeneration strategies. This study will enable future exploration of mechanisms through which ECs and EC-dependent paracrine factors stimulate salivary gland regeneration and facilitate future testing of these newly identified factors in salivary gland disease models for therapeutic benefit. Abbreviations: BM (bone marrow), DAPI (4',6-diamidino-2-phenylindole), EC (endothelial cell), FACS (fluorescent activated cell sorting), FGF2 (Fibroblast growth factor 2), FP (fluorescent protein), G-CSF (granulocyte colony-stimulating factor), IHC (immunohistochemistry), K (cytokeratin), MACS (magnetic bead activated cell sorting), MSC (mesenchymal stem cell), Mx-IHC (multiplexed immunohistochemistry), PEG (poly(ethylene glycol)), next generation RNA sequencing (RNA Seq), smooth muscle (SM), SMG (submandibular gland), SS (Sjogren's Syndrome), SCF(stem cell factor), VEGF164 (vascular endothelial growth factor, 164 aa)